Type I interferons form the frontline of innate host defenses by inducing an antiviral state in infected and neighboring cells, and by modulating adaptive immune responses directly and via the induction of interferon-stimulated genes (ISGs). Whereas ISGs are strongly induced during HCV infection, neither the ISG-inducing cytokines nor their cellular sources have been defined. This is important because the intrahepatic ISG level influences the response to antiviral therapy with interferon-based regimens. Whereas type I interferons are regarded as the major contributors to ISG induction in other viral infections, HCV has been shown to interfere with TLR3- and RIG-I-mediated induction of IFN-beta and with JAK/STAT signaling downstream of the IFN-alpha/beta receptor in infected cells, thus reducing IFN-alpha/beta production to levels that are undetectable in HCV-infected patients. In vitro studies suggest that plasmacytoid dendritic cells may be the source of the ISG-inducing type I interferons, but the role of pDCs has not been studied in the HCV-infected liver. In this context, type III interferons have become of interest. This family is composed of IL-29, IL-28A, and IL-28B and induced in response to several viral pathogens. Although signaling via the JAK-STAT pathway is shared with type I interferons and similar sets of ISGs are induced, receptors for type III interferons are distinct from those for type I interferons and expressed in a cell type-specific manner. In the liver, type III interferon receptors are expressed at significant levels as a functional, full-length form, suggesting intact type III interferon signaling as part of the intrahepatic innate immune response. Furthermore, single nucleotide polymorphisms (SNPs) near and within IL28B are strong predictive markers for spontaneous and treatment-induced HCV clearance, suggesting that variations in type III interferon expression or function affect the outcome of HCV infection. While recombinant type III interferons are known to suppress HCV replication in vitro, their expression level in the liver has never been studied prospectively during acute HCV infection. Thus, the relative antiviral effect of endogenously produced type I and type III interferons is not known. To identify the cytokines and cells that drive ISG induction and mediate antiviral activity during HCV infection we studied type I and III interferon responses in (i) serial non-human primate liver biopsies and plasma samples that were available from previous studies (Nat Med 2006, 12:190-197; Gastroenterology 2011;141:686-695) and (ii) primary human hepatocyte cultures (PHH) upon HCV infection. During the first 6 months of acute HCV infection type I interferons were minimally induced at the mRNA level in the liver and undetectable at the protein level in the plasma. In contrast, type III interferons, in particular IL-29 mRNA and protein, were strongly induced and these levels correlated with ISG expression and viremia. However, there was no association between intrahepatic or peripheral type III interferon levels and the outcome of acute HCV infection. The in vitro PHH model recapitulated the strong type III and weak type I interferon responses upon HCV infection. Supernatant of HCV-infected PHH mediated antiviral activity upon transfer to HCV-replicon containing cells, which was significantly blocked by neutralization of type III interferons and less by neutralization of type I IFNs. Furthermore, IL-29 production by HCV-infected PHH occurred independently from type I interferon signaling, and was not enhanced by the presence of plasmacytoid dendritic cells. The strong induction of IL-29, the main type III interferon in the acutely HCV-infected liver, may be attributed to a type I interferon-independent production, because neutralization of type I interferons did not affect IL-29 production in most of the tested PHH cultures and because IL-29 production was not further enhanced in the presence of plasmacytoid dendritic cells that secrete IFN-alpha in direct contact with HCV-infected PHH. Collectively, these results demonstrate that hepatocyte-derived type III interferons contribute to ISG induction and antiviral activity, and may therefore influence the response to interferon-based treatment regimens, but that they are not the principal determinant of the outcome of acute HCV infection.